The present invention concerns a new cosmetic composition for slimming containing L-arginine, an L-arginine analogue, or one of their derivatives, for topical application.
Cosmetic industry is continuously searching for new performing and really efficient active ingredients to fight localised adipose overloads.
Adipose tissue is mainly constituted of adipocytes, which are metabolically very active cells maintaining the energetic balance in the organism by two antagonist metabolic pathways:
lipogenesis, i.e. biosynthesis from glucose and fatty acids, of triglycerides stored into the adipocytes.
lipolysis, which is the hydrolysis of the triglycerides to glycerol and free fatty acids.
Amoung the slimming cosmetic products commercially available are compositions acting by inhibition of lipogenesis, containing inhibitors of glucose uptake or binding which is necessary for the triglycerides synthesis.
Other slimming products aim to stimulate lipolysis by acting on the AMPc level which stimulates the lipase activating the hydrolysis of triglycerides; most of these products contain xanthic bases, especially caffeine, usually associated with vegetal extracts which increase their activity. Some products contain active ingredients which have an action on xcex1 and xcex2 adrenergic receptors which respectively inhibit and stimulate lipolysis.
The influence of sexual hormones and catecholamines on xcex2-receptors was recently shown, but their use in cosmetic is forbidden.
Recent improvements in understanding the mechanisms of regulation of the adipocyte lead to consider new cosmetic strategies concerning slimming products for topical use.
The new approach described in this patent is the use of a topical lipolytic active ingredient acting on the adipocyte by inducing the release of a cellular mediator.
A researchers team has recently identified a potential regulator of lipolysis: the nitric oxide radical (NOo) or its related redox forms (Modulation of White Adipose Tissue Lipolysis by Nitric Oxide, Gaudiot et Coll., J.Biol. Chem., 1998, 273, 13475-13481). It arises from this work that, in an appropriate environment, NOo radical can act as a lipolysis stimulation factor.
Nevertheless, nitric oxide itself or a NO-donor use are not suitable for cosmetic applications.
Actually, nitric oxide radical has many physiologic properties because it is a cellular mediator acting particularly on vascular and neuronal systems.
Nitric oxide is also a potential cellular toxin involved in the immune response. In the skin, NOo takes part in the inflammatory response (for instance during photoinduced stress), modulates melanocytes activity or cellular proliferation in wound healing process.
Furthermore, in some cases, nitric oxide radical can generate in vivo toxic by-products such as peroxinitrous anion.
Thus, one of the aim of the present invention is to solve a part of the above mentioned problems providing a cosmetic composition for slimming, which contains a cosmetic active ingredient able to generate nitric oxide radical in an endogenous way.
L-arginine is the natural substrate of NO-synthase, the enzyme that metabolises arginine to release nitric oxide radical. This enzyme is naturally present in the adipose tissue and maintains a basal level of NOo.
Is was shown that an extracellular supply of L-arginine led to NOo production.
Although this enzyme is very specific, it was proved recently that it could accept other substrates, particularly an analogue of L-arginine, homo-L-arginine.
In the scope of this new cosmetic approach, the topical application of the active ingredient was taken in account, which raises the question of its bioavailability that is to say its ability to act on its main biological target: the adipocyte. This kind of cell is not localised in the direct vicinity of the active ingredient application area, but in a deep layer of the skin, hypodermis.
That is why an important aim of the invention is increasing the bioavailability of the cosmetic active ingredient according to the invention.
Thus, one of the characteristics of the cosmetic composition according to the invention is to reduce L-arginine (or its analogue) degradation (by arginases) or metabolisation in skin""s upperlayers, or to improve its cutaneous penetration.
NO-synthase is also present in the cells of the upperlayers of the skin, as keratinocytes and fibroblasts.
So an other characteristic of the invention is to avoid any possible side effects that may result from NOo production by skin""s upperlayers.
A positive consequence of NO-synthase presence in skin""s upperlayers is that the lipolytic composition containing NOo precursor will be able to produce a lipolytic signal which may be transmitted to a deeper layer as hypodermis.
Furthermore, endothelial cells, which form the wall of microvessels irrigating dermis and hypodermis, also have NO-synthases and may contribute to the lipolytic effect. Moreover, they can also help decreasing excess adipose tissue by a special mechanism: NOo can induce a vasodilatation which can promote adipose tissue""s draining.
At last, a possible mode of action of nitric oxide radical, suggested by new experimental data, could result from its ability to act against preadipocytes multiplication. Preadipocytes are precursor cells which differentiate in mature adipocytes, able to store fat. This property could fight very efficiently against adipose tissue increase.
The present invention consists in the use of L-arginine, an L-arginine annalogue, or one of their derivatives (or any of their salts) as an endogenous precursor of NOo, in cosmetic compositions for slimming with topical application.
So far, L-arginine, L-arginine analogue or their derivatives had never been used as NOo precursor for slimming.
The patent No FR-A-2.758.724 provides a composition against cellulite and adipose tissue""s excess, said composition comprising protamine, a protein containing among other aminoacids arginine. Thus, protamine may have an inhibitory action on fatty acids captation by adipocytes in the subcutaneous adipose tissue. Protamine may act by inhibition of lipogenesis, which is not the effect described in the present invention.
Arginine is also used in the patent No FR-A-2.745.498 which describes a therapeutic composition associating arginine and silicon used in high-protein low-calorie diets. This composition is orally administrable and aims to improve the effectiveness of a high-protein diet thanks to the lipolytic activity of silicon and to avoid side effects on kidney function with arginine. The patent No EP-A-0.281.435 relates to a therapeutic product consisting in asilanols-aminoacids complex, said product being appropriate to prepare medicines which are regulators and activators of metabolism, growth and multiplication of some cells. The active agent is the silanol, which biological activity is improved by complexation with a natural aminoacid that can be L-arginine.
Lastly, the use of L-arginine as NOo precursor was described, but the purpose was protecting the tissues from oxidative stress (Cordeiro P. G., Santamaria E, Hu Q. Y. xe2x80x9cUse of a nitric oxide precursor to protect pig myocutaneous flaps from ischemia-reperfusion injuryxe2x80x9dxe2x80x94Plastic and Reconstructive surgery, vol. 102, (1998) no6, pp. 2040-2048).
The present invention relates to a new cosmetic composition for slimming comprising, in association with any cosmetically acceptable excipient, at least one compound which is L-arginine, an L-arginine analogue or one of their derivatives, or any of their salts, said compound having the following general formula: 
where:
R1 represents a hydrogen atom, an hydroxyl group, an acyl or acyloxy radical, or an aminoacid substituted or not on its free xcex1-amino function, bound by a peptidic bond,
R2 represents an hydroxyl group, an amine, alkylamine or alcoxy radical, a silyloxy group, or an aminoacid substituted or not on its free xcex1-carboxylic function, bound by a peptidic bond,
and n is equal to 2 or 3
It is to be noted that the radicals R1 and R2 are biodegradable substituents of the xcex1-amino or xcex1-carboxylic function of L-arginine or its analogue, which can be hydrolyzed in vivo to the corresponding aminoacid.
Considering that L-arginine is the natural substrate of the enzyme NO-synthase, the stereochemistry of the asymmetric carbon of the L-arginine must be respected in all the compounds according to the invention, so that such compounds can be recognized as substrates by NO-synthase.
The compounds of formula (I) that are preferred are the compounds for which R1 represents a hydrogen atom and R2 represents a silyloxy group.
Preferably, the silyloxy group has the following general formula: 
where R3 represents an alkyl group.
In this case, a part of the silanols used to synthesize compound (I) can make a complex or combine by weak bonds with the NH2 group of L-arginine (or its analogue), which contribute to increase the stability of compound (I) and avoid its metabolisation in the upperlayers of the skin.
Among these compounds, a preferred compound is the monomethyl silanediol of L-arginine.
A preferred embodiment of the invention consists in reducing temporarily the polarity of L-arginine or its analogue, so as to favour the active ingredient""s penetration in epidermis.
This is simply realised by esterification of the xcex1-carboxylic acid function and/or amidification of the xcex1-amino function, which make these functions non ionizable at a physiologic pH.
The xcex1-carboxylic acid and xcex1-amino substitution by hydrophobic radicals contribute to increase the lipophilicity of the molecule and its cutaneous penetration.
The biodegradability of the substituents, i.e. their ability to hydrolyze at physiological pH or in the presence of cutaneous enzymes, is an important point of the invention because the compounds substituted in alpha position are not biologically active.
Preferably, the xcex1-amino function is substituted by an acetyl radical, or by a more hydrophobic substituent as tert-butyloxycarbonyl (Boc).
Preferably, the xcex1-carboxylic acid function is substituted by a radical R2 alcoxy or alkylamine, carrying an ethyl group or a more hydrophobic substituent as n-butyl, n-octyl radicals, or pivalloyloxymethyl, a substituent which is stable at the external pH of the skin (about pH 5) and hydrolyzed at a pH near to neutrality, that is the pH of the deeper skin""s layers.
According to a further preferred embodiment of the invention, the compound of general formula (I) is such as R1 and/or R2 is an aminoacid chosen among the following aminoacids alanine, cysteine, glycine, hydroxyproline, leucine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophane, valine, asparagine, glutamic acid, pyroglutamic acid, aspartic acid, glutamine, arginine, histidine, lysine.
Preferably, the aminoacid is alanine, leucine, isoleucine, phenylalanine, tryptophane or valine which are hydrophobic aminoacids able to increase the lipophilia of the compound in a way to augment its affinity for the skin.
When the radical R2 is an aminoacid, it is better to substitute its free xcex1-carboxylic acid function and/or the free xcex1-amino function of the aminoacid R1 to make this function non ionisable at physiological pH.
Preferably, these functions are substituted by the above mentioned groups.
As it was said, an important aim of the invention is to provide a NOo precursor, the L-arginine or its analogue, which biodisponibility is compatible with an action on hypodermis, a deep layer of the skin.
So it is useful to make a compound having a structure suitable to avoid its metabolisation into the skin""s upperlayers before it reaches hypodermis.
This is possible first by substitution of the xcex1-carboxylic acid and xcex1-amino functions of L-arginine. Thus, these forms are biologically inactive (prodrug concept) and will not enter into the metabolic pathways using this aminoacid.
These forms are not substrates of NO-synthases (Hrabak A, Bajor T, Temesi A xe2x80x9cComparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophagesxe2x80x9dxe2x80x94Biochem. Biophys. Res. Commun., vol. 198, (1994) pp. 206-212).
This resistance to enzymatic deactivation in skin""s upperlayers will also be reinforced when the substituents R1 and/or R2 carry a sterically hindering radical.
Thus, when R1 is an acyloxy radical and/or R2 is an alcoxy or alkylamine radical, the alkyl chain carried by these radicals can be an isopropyl, isobutyl, sec-butyl or tertiobutyl group.
Similarly, when R1 and/or R2 is an amino acid, the later is chosen in the group consisting of amino acids having a hindering side chain as valine, leucine or isoleucine.
Following an other embodiment increasing the resistance of compound (I) to enzymatic degradation, R1 and/or R2 radicals are chosen among aminoacids which are substrates of specific enzymes, so that said compound is less sensible to enzymatic deactivation when crossing the upperlayers of the skin.
Such aminoacids can be citrulline, homoserine, norvaline, ornithine, penicillamine or sarcosine.
According to this embodiment, a particularly interesting compound is the natural dipeptide L-citrullinyl-L-arginine (for which R1=citrulline and R2=OH) produced by a red alga (Chondrus crispus).
A particularly important aim of the invention, is providing a NOo precursor which do not induce embarrassing side effect, which is not acceptable for a cosmetic active ingredient.
Thus, as it has been specified, the cosmetic composition according to the invention is conceived in order to avoid NOo production by the skin""s upperlayers cells.
Keratinocytes or fibroblasts normally possess one form of NO-synthase called xe2x80x9cconstitutivexe2x80x9d, which produces NOo in tiny amounts (compatible with NOo""s cellular messenger role) and only in response to very specific stimuli.
However, in some pathologic conditions (specifically inflammation), these cells can express a form of NO-synthase called xe2x80x9cinduciblexe2x80x9d, able to produce larger amounts of NOo.
In this context, a particular embodiment of the invention aims to oppose to a toxic by-product of NO radical, peroxinitrous acid (HOONO), which can be produced when skin is submitted to an oxidative stress and which toxicity is increased in the presence of transition metal.
The principle consists in condensing the NOo precursor on a molecule able to counteract peroxinitrous acid toxicity; said molecule being released during xe2x80x9cbioactivationxe2x80x9d by cutaneous enzymes.
According to this particular embodiment, a biodegradable substituent R1 or R2, possessing a thiol (SH) or a thioether function, is condensed on L-arginine or its analogue.
In fact, the sulfur atom confers the ability to fix the metallic ions which participate to the formation of cytotoxic species derived from NO radical and therefore to neutralize them.
Moreover, it is known that in vivo, aminoacids possessing a thiol group, particularly cysteine, are preferred target for peroxinitrous acid.
So aminoacids possessing a thiol function chosen among L-cysteine, N-acetyl-cysteine, L-methinonine or penicyllamine will be preferably condensed on L-arginine or its analogue.
Other amino acids are also suitable to avoid possible side effects due to peroxinitrous acid formation, and particularly L-histidine which possesses an imidazole ring having the property to scavenge free radicals which can be generated by peroxinitrous acid (homolytic decomposition releasing OHo and NOo). Moreover, histidine is known for its chelating properties of transition metals which catalyses toxic reactions associated with peroxinitrous acid.
Advantageously, glutamic acid or aspartic acid can be chosen, which xcex1-carboxylic acid functions give them properties of chelation of transition metals.
Cosmetic compositions according to the invention can advantageously contain at least one compound having anti-oxidative properties in order to neutralize free radicals which can be generated by peroxinitrous acid, and to repare damages caused by this species (peroxidizing agent of cellular membranes).
Preferably, this anti-oxidative compound is a pseudo-dipeptide such as the type obtained by the coupling between histamine and an aminoacid and described in the international patent application WO94/19325.
An other particular embodiment of the invention consists in obtaining a composition containing a precursor permitting the endogenous synthesis of NOo while modifying favourably the redox state of the adipose tissue.
In fact, according to Gaudiot et coll., the lipolytic activity of nitrogen monoxide is modulated by its redox state (NOo, NO+ or NOxe2x88x92); it is particularly appropriated to create a redox state (tissular or intra-cellular) which is going to optimize the lipolytic activity of this messenger.
This can be obtained associating with the compound of general formula (I) an antioxidant as vitamin E (and others related tocopherols), vitamin C (ascorbic acid and derivatives), or a pseudo-dipeptide such as the above mentioned.
It can be advantageously condensed on L-arginine or its analogue a compound having antioxidative properties. Some of these compounds as aminoacids have been already mentioned but cysteine and preferably N-acetyl-cysteine are particularly indicated because their influence on the redox state of the cells has been described.
On the other hand, when the compound of general formula (I) is the monomethylsilanediol of L-arginine, the monomethylsilanediol released during the hydrolysis of the compound can also modulate the cellular redox state in order to maximize the lipolytic effect.
At least, insofar as the subject-matter of the invention consists in bringing to the skin a NO-synthase substrate able to induce endogenous formation of NOo, every additive favouring the activity of this enzyme will be suitable for this purpose.
It has been shown that substances bearing a reduced thiol were necessary for a maximal enzymatic activity.
Thus cystein and other thiol-containing compounds appear as valuable additives.
NADPH can be mentioned, which is a cofactor necessary for the enzyme""s activity and which is used during NO formation.
Thus, every substance contributing to the increase of NADPH level in the skin cells will be suitable to favour a NO-dependant lipolytic effect.
Similarly for calcium or every substance enable to increase the intra-cellular calcium level (this element is a cofactor of the enzyme too).
Therefore, monomethylsilanediol of L-arginine is one of the recommended compounds because monomethylsilanediol can favour calcium""s supply to the cells (osteoporose treatment).
The compounds of formula (I) can be prepared by methods known by one ordinary skilled in the art.
The cosmetic compositions according to the invention can be used under different forms suitable with cosmetics that is to say solution, emulsion or dispersion, or in the forms of liposomes, microparticles or nanoparticles or even on organic polymers matrix, silica or other mineral medium.
It will be preferably chosen forms enable to favour intradermal penetration as microemulsions, liposomes or Emulzome(copyright) which is a fluid microdispersion of fatty compounds/water commercialised by the applicant, having a high bioaffinity for the skin.
The compositions according to the invention can be in any form cosmetically appropriated, and especially in any form suitable for a topical use: cream, milk, lotion, gel, spray, patch.
All the additives and excipients commonly used in cosmetic industry are appropriate to carry out the invention provided they are compatible with compound of formula(I).
Among these additives and excipients can be mentioned: Transcutol(copyright) (monomethylic ether: of diethylene glycol), xcex1-Bisabolol, oleic acid or moreover urea. In addition to their specific properties, these last compounds favour the cutaneous penetration.